α-Amylases (E.C. 3.2.1.1) hydrolyze internal α-1,4-glycosidic bonds of starch and starch-like polymers with the formation of dextrins and β-1,6-branched oligosaccharides. They are very much among the industrially most important enzymes. Thus, for example, α-amylases are employed in the production of glucose syrup, for the treatment of raw materials in the manufacture of textiles, for the production of adhesives or for the production of sugar-containing food or food ingredients. Another important field of use is the use as an active component in detergents and cleansers.
Since detergents and cleansers have mainly alkaline pH values, particular use is made here of α-amylases that are active in alkaline medium. These types of α-amylases are produced and secreted by microorganisms, i.e. fungi or bacteria, especially those of the genera Aspergillus and Bacillus. Starting from these natural enzymes, there is now quite a vast abundance of variants available which have been derived via mutagenesis and have specific advantages depending on the field of use.
Examples thereof are the α-amylases of Bacillus licheniformis, B. amyloliquefaciens and B. stearothermophilus and their improved developments for the use in detergents and cleansers. The B. licheniformis enzyme is available from Novozymes under the name Termamyl® and from Genencor under the name Purastar® ST. Products of further development of this α-amylase can be obtained from Novozymes under the trade names Duramyl® and Termamyl® ultra, from Genencor under the name Purastar® OxAm and from Daiwa Seiko Inc., Tokyo, Japan, as Keistase®. The B. amyloliquefaciens α-amylase is sold by Novozymes under the name BAN®, and variants derived from the B. stearothermophilus α-amylase are sold under the names BSG® and Novamyl®, likewise by Novozymes.
Examples of α-amylases from other organisms are further developments of the Aspergillus niger and A. oryzae α-amylases, obtainable under the trade name Fungamyl® from Novozymes. Another commercial product is Amylase-LT®, for example.
Mention may further be made of the Bacillus sp. A 7-7 (DSM 12368) α-amylase disclosed in the application WO 02/10356 A2 and of the B. agaradherens (DSM 9948) cyclodextrin glucanotransferase (CGTase) described in the application WO 02/44350 A2. In addition, for example, the applications WO 03/002711 A2 and WO 03/054177 A2 define sequence spaces of α-amylases, all of which could be suitable in principle for corresponding applications.
The application DE 10309803.8, which has not been prepublished, for example, describes point mutations for improving the activity of said enzymes in alkaline medium. According to this application, amino acid substitutions suitable here are those in positions 13, 32, 194, 203, 230, 297, 356, 406, 414 and/or 474, according to the numbering of the unprocessed B. amyloliquefaciens α-amylase.—These positions are according to the numbering of the unprocessed Bacillus sp. A 7-7 (DSM 12368) α-amylase (WO 02/10356 A2) L13, T36, W198, S201, 1208, A235, D302, D361, H408, K416 and N476, respectively, with the following, particularly effective substitutions: 13P, 32A, 194R, 197P, 203L, 230V, 297D, 356D, 406R, 414S and 474Q.
Another example of point mutagenesis on α-amylases is the application WO 00/22103 A1 which discloses polypeptides, inter alia also α-amylase variants, containing mutagenized surface amino acids. The purpose of this mutagenesis was to reduce the immunogenicity and/or allergenicity caused by these molecules.
Fusion products of α-amylases for the use in detergents and cleansers have also been described. Thus, for example, the application WO 96/23874 A1 discloses hybrids of the α-amylases of Bacillus licheniformis, B. amyloliquefaciens and B. stearothermophilus. According to the teaching of this application, such hybrid amylases may be prepared for determining the three-dimensional structure of said amylases, in order to use said structure for detecting important positions for enzymic activity. Further developments in this respect are the applications WO 97/41213 A1 and WO 00/60059 A2, which report numerous α-amylase variants whose respective performances have been improved. The application WO 03/014358 A2 discloses special hybrid amylases of B. licheniformis and B. amyloliquefaciens. 
The three applications WO 96/23873 A1, WO 00/60060 A2 and WO 01/66712 A2, which are the basis of the commercial product Stainzyme® from Novozymes, constitute another important prior art. All the variants obtainable by point mutagenesis which are specified in each of these applications have altered enzymic properties and are therefore claimed or described for the use in detergents and cleansers. WO 96/23873 A1 makes mention of, in some cases two or more, point mutations in more than 30 different positions in four different wild type amylases. They apparently have altered enzymic properties with regard to thermal stability, oxidation stability and calcium dependence. They include point mutations in the following positions, each of which is stated with respect to the Bacillus sp. NCIB 12512 α-amylase: substitution of oxidizable amino acids in positions M9, M10, M105, M202, M208, M261, M309, M382, M430 or M440, preferably M91L; M10L; M105L; M202L,T,F,I,V; M208L; M261L; M309L; M382L; M430L and M440L; deletions of F180, R181, G182, T183, G184 and/or K185; and additionally the substitutions K269R; P260E; R124P; M105F,I,L,V; M208F,W,Y; L217I; V206I,L,F; Y243F; K108R; K179R; K239R; K242R; K269R; D163N; D188N; D192N; D199N; D205N; D207N; D209N; E190Q; E194Q or N106D.
The application WO 00/60060 A2 likewise specifies a multiplicity of possible amino acid substitutions in 10 different positions, on the basis of two very similar α-amylases from two different microorganisms, with the same numbering α-amylases AA349 and AA4560). These are the following sequence variations: R181*, G182*, D183*, G184*; N195A, R, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; 1206A, R, D, N, C, E, Q, G, H, L, K, M, F, P, S, T, W, Y, V; E212A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; E216A, R, D, N, C, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V; K269A, R, D, N, C, E, Q, G, H, I, L, M, F, P, S, T, W, Y, V; and/or R181A, N, D, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, V. This development too is against the background of improving performance via mutations.
WO 01/66712 A2, finally, refers to 31 different amino acid positions which are partially identical to the ones mentioned above and which have been mutated in either of the two α-amylases specified in the application WO 00/60060 A2 and which are said to improve aspects of both performance and stability. These are point mutations in the following positions: R28, R118, N174; R181, G182, D183, G184, G186, W189, N195, M202, Y298, N299, K302, S303, N306, R310, N314; R320, H324, E345, Y396, R400, W439, R444, N445, K446, Q449, R458, N471 and N484, again as defined via α-amylase AA560, i.e. also according to the numbering of the letter. Among these, the following variants are said to be particularly advantageous: Delta G184; Delta (R181-G182); Delta (D183-G184); R28N,K; S94K; R118K; N125A,R,K; N174D; R181Q,E,K; G186R; W189R, K; N195F; M202L,T; Y298H,F; N299A; K302R, S303Q, N306G,D,R,K; R310A,K,Q,E,H,D,N; N314D; R320K; H324K; E345R,D,K,N; Y396F; R400T,K; W439R; R444K; N445K,Q; K446N; Q449E; R458K; N471E and N484Q.
There is a very extensive prior art available on the improvement in the stability of α-amylases to very different influences by carrying out point mutations on the molecules in question. Said prior art will be treated here to an appropriate extent, taking into account only the documents directed to the enzymes and substantially dispensing with those documents which describe again the use of the (previously described) enzymes in question in a large variety of industrial sectors:
The applications WO 91/00343 A2 and EP 409299 A2 describe variants of B. amyloliquefaciens α-amylase which have reduced stability and substitutions in, in some cases two or more of, positions 113, 114, 116, 123, 163, 164, 166, 238, 316, 322, 345, 349, 356, 386, 394 and 398, including the particularly advantageous variant in which R123 has been replaced with other amino acids, in particular C.
The application WO 91/00353 A2 claims all α-amylase variants having increased thermostability, in which amino acid substitutions have been performed in positions 111, 133 and/or 149 (according to the numbering of B. licheniformis α-amylase); they include the specifically described substitutions A111T, H133Y and T1491 which are said to be advantageous.
The application WO 94/02597 A1 claims all α-amylase variants in which one or more methionine (M) residues have been replaced with, in principle, any other amino acid except C and M; among these, the newly to be inserted amino acids L, T, A, G, S, I or D, particularly L, T, A or G, are said to be advantageous; this is said to apply particularly to the M residues in positions 197, 200 and 206 (according to the numbering of B. licheniformis α-amylase, i.e. termamyl), with the termamyl variants of A, G, T, V, Q, F, L, H, I, S, N, C and D in position 197 being specifically described.
The application WO 94/18314 A1 claims α-amylase variants stabilized to oxidation which are obtained by deleting methionine (M), tryptophan (W), cysteine (C) or tyrosine (Y) residues of the precursor molecule or substituting other amino acids for said residues; among these, positions M8, M15, M197, M256, M304, M366 and M438 (according to the numbering of B. licheniformis α-amylase) are emphasized and the variants M197A, I, T, C, M15L, T, N, D, S, V, I and any variants of W138, including deletion thereof, are explicitly claimed. The substitutions of any possible amino acids for M8A, M15 and M197, in particular M197L, M256A, M304L, M366 A, Y, M438A and W138F, Y, A, L, H and C, are specifically described.
The application WO 96/05295 A2 reveals corresponding variants, in particular those containing the amino acid substitutions M15T, M197T, M15S, W138Y, for use in bleach-containing agents, partly in combination with one another.
U.S. Pat. No. 5,824,532 specifies the substitutions T, L, A, R, N, D, E, G, I, K, F, P, S, V, H, Q for M197 (according to the numbering of B. licheniformis α-amylase) or deletion thereof and also the substitutions M15 L, T, N, D, S and V as those which can be used for stabilizing α-amylases to oxidation; in addition, a substitution of W138 is also recommended here.
U.S. Pat. No. 6,297,037 B1, of the same family, discloses among these the substitutions M197T, M15L, M15T, W138F and W138Y and combinations thereof as particularly stable to oxidation, temperature and/or alkali.
The application WO 95/10603 A1 claims in principle any α-amylase variants having improved washing or cleaning performance, on which a deletion, substitution or addition of a single amino acid has been performed, except M197A or M197T (according to the numbering of B. licheniformis α-amylase) as sole modification. Among these, the claims emphasize variants in positions 1, 2, 3, 15, 17-35, 29-35, 51-58, 68, 85, 88, 94-104, 121-136, 140, 142, 148, 152, 142-182, 172-178, 187, 209, 217, 230, 233, 242, 246-251, 255, 260-269, 290-293, 314-320, 341, 360, 369-383, 393, 398, 409, 416-421, 435, 450, 458-465. Of these, only the following modifications are described experimentally, partially in multiple combinations with one another: 1*, 2*, L3V, M15T, R23K, S29A, A30E, Y31H, A33S, E34D, H351, H68Q, S148N, M197I, N, S, T; A209V, L230I, V233A, R242P, E255P, T341P, S373P, Q374P, H450Y, E458D, P459T, V461K, N463G and E465D.
The application WO 95/35382 A2 claims any α-amylase variants having a similarity of at least 70% identity to B. licheniformis α-amylase, in which variants amino acids in positions 104, 128, 187 and/or 188 (according to the numbering of said α-amylase) have been replaced with any other amino acids. Among these variants, the substitutions N104D, V128E, S187D and N188D are particularly emphasized because they are described in combination with one another and/or with other substitutions disclosed in the prior art as those which improve the enzymic properties regarding various aspects.
The application WO 96/23873 A1 describes variants of four different α-amylases having properties, including also regarding stability, which have been altered compared with the starting enzyme. Among them those variants are claimed which have a reduced sensitivity to oxidation due to an M, W, C or T having been replaced with a less oxidizable amino acid. Specific mention is made here of the residues M9, M10, M105, M202, M208, M261, M309, M382, M430 and M440 (according to the numbering of B. licheniformis α-amylase), in particular in the substitution by L, and the substitutions M202L, T, F, I, V.
The application WO 96/23874 A1 discloses numerous variants obtainable by point mutations. These include those having point mutations (in each case according to the numbering of B. licheniformis α-amylase) in positions 236 (N236I, Y, F, L, V), 281 (H281F, I, L) and 273 (Y273F, W), which have relatively high activity at relatively high pH, and those having point mutations in positions 61, 62, 67, 106, 145, 212, 151, 214, 150, 143, 146, 241, 236, 7, 259, 284, 350, 343, 427 and 481 (L61W, V, F; Y62W; F67W; K106R, F, W; G145F, W; I212F, L, W, Y, R, K; S151 replaced with any other amino acid, particularly F, W, I or L; R214W; Y150R, K; F143F; R146W; L241I, F, Y, W; I236L, F, W, Y; L7F, I, W; V259F, I, L; F284W; F350W; F343W; L427F, L, W and V481F, I, L, W), which have increased thermostability and/or elevated temperature optimum.
As a development of WO 96/23874 A1, the application WO 97/41213 A1 claims any variants of α-amylases characterized by their similarity to that of B. licheniformis and having the following mutations (in each case according to the numbering of B. licheniformis α-amylase): A181E,D,Q,N,V; I201 (replaced with a bulkier amino acid), including I201W,F,L; Y203Q; Q9K,L,E; F11R,K,E; E12Q; D100N,L; V101H,R,K,D,E,F; V102A,T; I103H,K; N104R,K,D; H105R,K,D,E,W,F; L196R,K,D,E,F,Y; I212R,K,D,E; L230H,K,I; A232G,H,F,S,V; V233D; K234L,E; I236R,K,N,H,D,E; L241R,K,D,E,F; A260S; W263H; Q264R,D,K,E; N265K,R,D; A269R,K,D,E; L270R,K,H,D,E; V283H,D; F284H; D285N,L; V286R,K,H,D,E; Y290R,E,K; V312R,K,D,E; F323H; D325N, N326K,H,D,L; H327Q,N,E,D,F; Q330L,E; G332D; Q333R,K,H,EL; S334A,V,T,L,I,D; L335G,A,S,T,N; E336R+R375E; T337D,K; T338D,E; T339D; Q360K,R,E; D365N; or G371D,R; having substitutions in positions H68, H91, H247, R305, K306, H382, K389, H405, H406, H450 or R483; having the following mutations: H140Y; H142Y; H159Y; H140D+H142R; H140K+H142D; or H142Y+H156Y; having the deletion of three amino acids in the subsequence from T369 to I377; having the substitution of the subsequence from T369 to I377 by any of the following partial sequences: I-P-T-H-S-V; I-P-T-H-G-V; I-P-Q-Y-N-I; having substitutions in positions R169 or R173, including R169I,L,F,T or R173I,L,F,T; having the mutations H156D; I201F; I212F; A209L,T; or V2081; having substitutions in positions N172, A181, N188, N190, H205, D207, A209, A210, E211, Q264 or N265, including N172R,H,K; A181T; N188P; N190L,F; H205C; D207Y; A209L,T,V; A210S; E211Q; Q264A,E,L,K,S,T; N265A,S,T,Y; or Q264S+N265Y or H156Y+A181T+A209V; or in the form of further special multiple variants comprising at least five amino acid substitutions. In each case, some of these variants are said to have altered enzymic properties, with inter alia also increased stability to deviating pH, elevated temperatures and oxidation being mentioned.
The application WO 98/26078 A1 describes stabilizing substitutions or deletions in the B. licheniformis α-amylases, that is in positions A33, A52, S85, N96, H133, S148, A209, A269, A379 and A435 (according to the numbering of B. licheniformis α-amylase), with the following variants being specifically referred to: A33S, A52S, N96Q, H133Y, S148N, A209V, A269K, A379S and A435S. Among these, the substitutions A33S, A52S, N96Q, H133Y, S148N, A209V and A379S are described experimentally, in various combinations with further substitutions, namely M15T, D28N, N188S, A210S, T322A, G433D and I479T.
The European application EP 985731 A1 derived from the international application WO 98/44126 A1 describes the α-amylase from B. species KSM-AP 1378, which has been modified in position 202; particular mention is made of the substitutions M202T, I, L, A, V, S, C. Accordingly, these variants have improved activity in the alkaline region and higher stability to oxidation. In contrast, the substitutions M9L, M105L (or I), M116D, M382L, M430L (or I) and M208Y are said to have had no increased stability over the wild type molecule.
The application WO 99/02702 A1 claims α-amylase variants in which, for the purpose of stabilization, an additional cystine bridge has been introduced by way of a point mutation; the amino acid substitutions E119C/S130C and/or D124C/R127C (according to the numbering of B. licheniformis α-amylase) are said to be advantageous to this end, and these modifications can apparently be combined with variations in other positions.
The application WO 99/09183 A1 describes and claims α-amylase variants in which the amino acids A210, H405 and/or T412 (according to the numbering of B. licheniformis α-amylase) have been replaced with other ones. Of these variants, the B. licheniformis α-amylase variant A210T/H405D/T412A is described in more detail.
The application WO 99/19467 A1 claims any α-amylase variants in Which up to six of the following deletions or substitutions have been carried out (according to the numbering of α-amylase S707(SEQ ID NO. 3)): (1.) R181*, G182*, T183*, G184*; (2.) N195, replaced with any other amino acid; (3.) V206, replaced with any other amino acid; (4.) E212, replaced with any other amino acid; (5.) E216, replaced with any other amino acid and/or (6.) K269, replaced with any other amino acid; they are said to have altered enzymic properties, including also improved stability. Variants which are particularly stable to acid and calcium-independent and which are distinguished experimentally, are N190F/Q264S of B. licheniformis α-amylase and I181*/G182*, I181*/G182*/N193F and I181*/G182*/N193F/E214Q of B. stearothermophilus α-amylase.
The application WO 99/23211 A1 claims any α-amylase variants in which amino acids in positions 141, 142, 143, 144, 145, 146, 147, 148, 149, 174, 181, 182, 183, 184, 185, 186, 189, 193, 195, 107, 108, 109, 166, 167, 168, 169, 170, 171, 172, 173, 267, 268, 269, 270, 271, 272, 273, 274, 275, 311, 346, 385, 456, 457, 458, 459, 460, 461, 462 and/or 463 (according to the numbering of B. licheniformis α-amylase SP722) have been replaced with various other ones or have been deleted. Among these, the following point mutations are emphasized: K142R; S193P; N195F; K269R,Q; N270Y,R,D; K311R; E346Q, K385R; K458R; P459T; T461P; Q174*; R181Q,N,S; G182T,S,N; D183*; G184*; K185A,R,D,C,E,Q,G,H,I,L,M,N,F,P,S,T,W,Y,V; A186T,S,N,I,V,R; W189T,S,N,Q. These are said to have improved properties, inter alia with respect to stability to temperatures of between 10 and 60° C. and/or pH of between 8 and 10.5.
The application WO 00/29560 A1 reveals α-amylase variants, in particular of hybrid amylases having point mutations in one or more positions on the surface, whereby overall hydrophobicity is increased or the number of side-chain methyl groups exposed to the medium are increased. This is said to increase thermostability. The residues E376, S417, A420, S356 and Y358 (according to the numbering of B. licheniformis α-amylase) are referred to as preferred targets to this end, with the substitutions E376K, S417T, A420Q, R; S356A and/or Y358F being preferred. They may additionally be combined with substitutions in positions K176, I201 and/or H205, preferably K176R, I201F and/or H205N.
The application WO 01/34784 A1 describes variants of fungamyl (Aspergillus oryzae) α-amylase which per se has only low thermostability, and claims any α-amylases which are at least 60% identical to Fungamyl and which according to this are understood as being a different group than the Termamyl-like α-amylases. The substitutions are said to be in the regions 98-110, 150-160, 161-167, 280-288, 448-455 and 468-475 (according to the numbering of A. oryzae α-amylase); of these, only the substitution Q153S is experimentally demonstrated to be stabilizing.
The application WO 02/31124 A2 specifies numerous point mutations obtained by random mutagenesis of B. sp. KSM-K38 α-amylase. They are said to change the properties of the molecule, with the particular influences not being named in detail. The substitutions are as follows (according to the numbering of said amylase), it being possible for a plurality of substitutions also to occur together: G2P,A; M9I,L,F; H14Y; L15M,I,F,T; E16P; H26Y,Q,R,N; D27N,S,T; G48A,V,S,T; N49X; Q51X; A52X; D53E,Q,R; V54X; A58V,L,I,F; V73L,I,F; E84Q; G88X; D94X; N96Q; M103I,L,F; N104D; M/L107G,A,V,T,S,I,L,F; G108A; F111G,A,V,I,L,T; A114D,I,L,M,V,R; T125S; D128T,E; S130T,C; Y133F,H; W138F,Y; G140H,R,K,D,N; D142H,R,K,N; S144P; N148S; A149I; R156H,K,D,N; N161X; W165R; D166E; R168P; E171L,I,F; H173R,K,L; I173L; L174I,F; A178N,Q,R,K,H; N179G,A,T,S; T180N,Q,R,K,H; N181X; N183X; W184R,K; D187N,S,T; E188P,T,I,S; N190F; D194X; L197X; G198X; S199X; N200X; I201L,M,F,Y; D202X; F203L,I,F,M; S204X; H205X; E207Y,R; Q209V,L,I,F,M; E210X; E211Q; L212I,F; D214N,R,K,H; D221N; E222Q,T; D224N,Q; Y228F; L230I,F; I233A,V,L,F; K234N,Q; P237X; W239X; T241L,I,F,M; S242P,R; A252T; D253G,A,V,N; Q254K; D255N,Q,E,P; G260A; K264Q,S,T; D265N,Y; V267L,I,F,M; D275N,T; E276K; M277T,I,L,F; E280N,T,Q,S; M281H,I,L,F; V286X, preferably V286Y,L,I,F; Y290X; Y293H,F; S301G,A,D,K,E,R; R305A,K,Q,E,H,D,N; E314K,Q,R,S,T,H,N; A315K,R,S; I318L,M,F; T329S; E333Q; A340R,K,N,D,Q,E; D341P,T,S,Q,N; G356Q,E,S,T,A; S375P; A376S; K377L,I,F,M; M3801L,F; E383P,Q; L384I,F; D386N,Q,R,K,I,L; Q389K,R; Y399A,D,H; W403X; D404N; I405L,F; V406I,L,F,A,D; N427X; H441K,N,D,Q,E; R442Q; Q444E,K,R; A445V; Q448A; H453R,K,Q,N; A454S,T,P; G472R, N479Q,K,R; Q480K,R. Experimentally, however, only the following variants are disclosed: E84Q, N96D, A315S, A445V, G464N, N121D and N390H; further mention is made of the possibility of performing the substitutions T125S, S144P, I173L, D210E, N393H, V408I, R442Q, N444H, Q448A and G464S. Specific effects are not demonstrated.
All of these applications regarding point mutagenesis share the fact that the point of stability is also evaluated under the aspect of a good performance in the corresponding field of use. This is because the stability maintained during storage and usage of α-amylases, for example within the context of detergent formulations, results in a high performance or in a performance as constantly high as possible with corresponding usage. They do not include any documents which specifically relate to the increase in stability during preparation, purification and formulation, i.e. processing of said α-amylases.
The purpose of increasing stability is also pursued by numerous applications describing special enzyme stabilizers. These additional ingredients cause a protein and/or enzyme present in corresponding agents to be protected from damage such as, for example, inactivation, denaturation or decay, particularly during storage. Thus, reversible protease inhibitors form a group of stabilizers. Others, for example polyols, stabilize to physical influences such as freezing, for example. Other polymeric compounds such as acrylic polymers and/or polyamides stabilize the enzyme preparation inter alia to pH fluctuations. Reducing agents and antioxidants increase stability of the enzymes to oxidative decay.
Compounds of these kind are added to the enzymes both during application and in the course of their work-up, which is particularly important, if a previously present stabilizer has been removed together with the other contaminations in a component step of said workup, for example a precipitation.
The prior art regarding the improvement in stability of α-amylases can be summarized as follows: a multiplicity of α-amylase variants have been developed via point mutagenesis, with the aim of these developments having mainly been that of improving the performance of said α-amylase variants. This category also includes those variants which have been stabilized with regard to denaturing agents such as bleaches or surfactants, since in these cases, the desired performance of the enzyme is optimized. In other cases, additional compounds which are overall referred to as stabilizers are mainly used for increasing stability or maintaining the physicochemical conformation.
A previously less regarded aspect in enzyme development is that of stabilizing the molecules per se in such a way that they have increased stability over the wild type molecule even during their workup. An additional advantageous effect thereof would be that this increased stability should also benefit the intended later usage of the enzyme in question.
The necessity for this is particularly evident in the case of α-amylases. At least some of these exhibit marked instability, especially during production and workup, which manifests itself macroscopically in a loss of activity. This applies in particular to process steps during which they are in aqueous solution, in particular at elevated temperature and at high (more alkaline) pH. As a result, the activities in question are lost even during workup. The work-up process includes all steps of industrial production, starting from isolating the enzyme in question, in particular the fermentation media common in biotechnological production, via the following washing and separation steps (for example by precipitation) and concentration up to formulation, for example granulation. However, the loss of activity may also occur during storage of α-amylase-containing agents or during application, for example when used as active ingredient in washing or cleaning processes.
This problem can go as far as individual α-amylases, although they can be produced and studied on a laboratory scale, refusing to be produced on an industrial scale with the aid of generally common methods. This is then also referred to as said enzymes having low process stability or processivity, meaning a large variety of possible processings and uses. For example, Bacillus sp. A 7-7 (DSM 12368) α-amylase exhibits greater instability than the native B. licheniformis α-amylase. Approaches to eliminate said instability would only enable such enzymes in the first place to be accessible to production on an industrial scale and thus to the large variety of fields of use in industrially relevant quantities.